Hitherto, production of foreign proteins utilizing genetic recombination technology has been extensively conducted by using microorganisms such as Escherichia coli, Saccharomyces cerevisiae or Bacillus, animal cells, plant cells and insect cells. As such foreign proteins, various biogenic proteins are considered to be accessible, and many of them have been industrially produced by using these living organisms for medical use so far.
However, methods employing procaryotes are not effective for all polypeptides, and it is not always easy to reproduce the complicated post-translational modification of eucaryotic proteins and to reproduce the natural steric structures. In addition, Escherichia coli has a characteristic endotoxin, which might contaminate end products. On the other hand, as for methods employing animal, plant or insect cells, these cells are more difficult to handle than microorganisms, their culture is costly, and production efficiency is low. For this reason, yeasts, eucaryotic microorganisms, are considered as the best for production of foreign proteins, especially eucaryotic proteins. Their culture methods are well established, and they do not contain endotoxins. Therefore, expression vectors for use in various yeast hosts have been developed so far (Romanos, M. A. et al., Yeast 8, 423-488, 1992).
Among various yeasts, S. pombe is considered to be closer to higher animals in various properties such as cell cycle, chromosomal structure and RNA splicing than other yeasts inclusive of Saccharomyces cerevisiae. The post-translational modification such as acetylation, phosphorylation and glycosylation of proteins produced in S. pombe seems fairly similar to that in animal cells (Russell, P. R. and Nurse, P., Cell 45, 781-782, 1986; Kaufer, N. F. et al, Nature 318, 78-80, 1985; Chappell, T. G. and Warren, G., J. Cell. Biol. 109, 2693-2702, 1989). Therefore, use of S. pombe as a host for expression of a foreign protein is expected to provide a gene product closer to its natural form, like that produced by animal cells. Since yeasts have a lot of commonness in their culture methods, knowledges about other yeasts can be easily applied to the yeast. Therefore, it is obviously advantageous to use S. pombe for production of a foreign protein by using microbiological methods and the DNA recombination technique.
However, S. pombe is far behind Escherichia coli and Saccharomyces cerevisiae in studies on genetic recombination using them. Especially, with respect to gene expression in S. pombe, only a small number of studies have been reported (Japanese Unexamined Patent Publications Nos. 181397/1986, 283288/1990 and 63596/1992). This is because development of expression vectors which have powerful promoters, are stable in S. pombe cells and are suitable and convenient for introduction of a gene has been retarded. Recent development of vectors for the fission yeast with a high expressivity which contains an animal virus-derived promoter region eventually opened the way to mass production of foreign proteins by S. pombe (Japanese Unexamined Patent Publications Nos. 15380/1993 and 163373/1995, which disclose inventions of the present inventors). This technique enabled many intracellular proteins to be produced easily and therefore is fairly useful.
Production of (foreign) eucaryotic secretory proteins by yeasts scarcely succeeded so far because yeasts can hardly recognize inherent signal sequences of foreign secretory proteins and therefore can not secrete the products from the cells into culture media. Further, at the time of purification, it was necessary that after cell rupture, the desired protein should be isolated from various coexistent cell components to avoid inactivation. Secretory production of a foreign protein is not only preferable in view of the easiness of purification, but also advantageous in that the product is identical or fairly similar to its naturally occurring counterpart in steric structure, because the protein to be secreted enters the secretory pathway in the host cells and undergoes appropriate processings such as formation of disulfide bonds and glycosylation.
However, few signal sequences that effectively function in the fission yeast have been reported (Tokunaga, M. et al., Yeast 9, 379-387, 1993; Broker, M. et al., B.B.A. 908, 203-213, 1987), no secretory expression vectors have been practically developed. On the other hand, the present inventors studied P-factor, which is a protein secreted by S. pombe from the cells and involved in mating as a mating pheromone. As a result, they found the fact that after conversion from its precursor by various enzymes in S. pombe, P-factor is secreted into a culture medium. They also determined the amino acid sequence and the gene of the P-factor precursor (Imai, Y. and Yamamoto, M., Gene & Dev. 8, 328-338; Japanese Unexamined Patent Publication No. 327481/1994). The amino acid sequence of the P-factor precursor is SEQ ID NO: 1 in the Sequence Listing give afterwards.